Steven Fussner
12/08/09
BIO 464 Lab
Bradley University
Tetrahymena thermophila Cyclin THERM_00180970 (CYC3)
Abstract:
The sexual life cycle in Tetrahymena thermophila is called conjugation. Conjugation can
be induced by stressing two different cell mating types and then pairing them in order to promote
exchange of haploid gamete nuclei. The result of conjugation is two daughter cells with a
transcriptionally silent micronucleus and a transcriptionally active macronucleus. The cyclin
family proteins are important in regulation of the cell life cycle. Expression data from a public
database for the cyclin gene TTHERM_00180970 (CYC3) was obtained to help determine which
steps of the conjugation cycles the cyclin protein is involved in regulating. RT-PCR analysis
was done to complement this data. During the sexual life cycle TTHERM_00180970 was
expressed at specific time throughout conjugation. The data was then compared to other studies
on expression of the specific gene. TTHERM_00180970 was observed to be expressed
vegetatively and with an increase in expression at C1, C6 and C13. The role of the cyclin protein
TTHERM_00180970 (Cyc3) was deduced to be involved in stabilization of the macronucleus
from its expression pattern during vegetation and conjugation and the known cellular events
involved in these processes.
Introduction:
Tetrahymena thermophila is a ciliate protozoa that is used as a model organism in
biological research. Tetrahymena is a nuclear dimorphic organism that undergoes conjugation
when stressed to exchange genetic information. When conjugation begins, two different mating
types will undergo meiosis, select a haploid nucleus and then exchange it with the conjugated
cell. The cells will then undergo two postzygotic nuclear divisions at which point a micro and
macronucleus will be selected. The resulting micronucleus will be transcriptional silent, while
the macronucleus will be heavily modified to increase its transcriptional abilities. The old
macronucleus will be degraded and each cell will undergo cell division producing two daughter
cells that each contain a silent micronucleus and a modified macronucleus (Miao et al. 2009).
The nuclear and cellular divisions involved in meiosis and mitosis are regulated by a
family of proteins called cyclins. Cyclins family proteins work by both promoting and halting
cell specific stages by binding and activating cyclin dependent kinases (CDKs). CDKs can then
phosphorylated other proteins required for the particular stage and either activate or inactivate
them. Once the specific cell stage is complete the cyclin will be degraded and the cyclin for the
next cell stage will be expressed (Zhang et al. 1999)
The role of specific cyclins in Tetrahymena thermophalia conjugation has not been
thoroughly investigated and was the focus of our research. TTHERM_00180970 was determined
to be a cyclin family protein by a BLAST search of the amino acid sequence which showed it to
have a cyclin homology domain. From this we looked into the expression patterns through an 18
hour mating cycle.
Figure 1: Tetrahymena conjugation cycle (Miao et al., 2009)
The cyclin coded by the gene TTHERM_00180970 (CYC3) has been shown to be
expressed during normal growth stages of Tetrahymena followed by a decrease in expression
upon starving and time zero of mixing mating types. In our analysis of TTHERM_00180970
expression we showed a sharp increase at the first hour followed by little to no expression for
hours two through six which is consistent with the expression profile on the Tetrahymena
Genome Database (TGDB) except for the expression at hour one. TTHERM_00180970 was
shown to be actively transcribed from hours seven through ten with it peaking around hour 8
based on the data provided from TGDB. At hour 10, expression is near normal growth values
and slowly increases throughout the rest of the conjugation cycle.
Figure 2: For growing cells, L-l, L-m and L-h correspond respectively to ~1x105 cells/ml, ~3.5x105 cells/ml and ~1x106
5 cells/ml were collected at 0, 3, 6, 9, 12, 15 and 24 hours（referred to as S-0, S-3, S-6,
cells/ml. For starvation,
S-9, S-12, S-15 and S-24). For conjugation, equal volumes of B2086 and CU428 cells were mixed, and samples were
collected at 0, 2, 4, 6, 8, 10, 12, 14, 16 and 18 hours after mixing (referred to as C-0, C-2, C-4, C-6, C-8, C-10, C-12, C14, C-16 and C-18). ( http://tged.ihb.ac.cn/search.aspx?keyword=TTHERM_00180970 )
Methods:
Cyclin genes were identified at the Tetrahymena Genome Database (www.ciliate.org) by
searching for proteins with the keyword “cyclin”. A BLAST search with a cyclin protein
sequence ensured that all cyclin genes were identified using this method. Microarray data during
conjugation (Miao, et al., PLoS ONE. 2009; 4(2): e4429) were collected for each gene from the
Tetrahymena Gene Expression Database (TGED; http://tged.ihb.ac.cn). PCR primers flanking
an intron were generated for each gene using Primer3 (Steve Rozen and Helen J. Skaletsky 2000)
and ordered from Integrated DNA Technology (Coralville, IA). The forward primer
TTHERM_00180970cDNA-F was (5’-CATTGAAGAGAGAATTTCCCACA-3’; Tm: 53.0°C)
and the reverse primer TTHERM_00180970cDNA-R was (5’-GGCTGCTTTCACTTCTTTCC3’; Tm 54.3°C). Oligo-dT-primed M-MLV reverse transcription (RT; Ambion) was performed
on RNA collected from control cells and from cells at various stages of conjugation using the
Trizol reagent (Invitrogen) according to the manufacturer’s protocol. 1 mL of cells (2.1 x 103
cells/mL) was collected at each time point, pelleted at 6k rpm, supernatant discarded, and cells
resuspended in 1 mL of Trizol. 180 ng of each template RNA was used per reverse transcription
reaction. cDNA was diluted 1:5 and used as a template for PCR. PCR was performed in 25 uL
reactions using GOTaq (Fisher, Hampton, NH) with 1 uL of each primer (10 uM). 15 uL of
completed PCR reaction products were separated on a 2% agarose gel. DNA bands were
visualized using ethidium bromide and photographed with a Kodak EDAS290 imaging system.
Band intensities were determined using ImageJ (Abramoff, M.D., Magelhaes, P.J., Ram, S.J.
"Image Processing with ImageJ". Biophotonics International, volume 11, issue 7, pp. 36-42,
2004).
Results:
RT-PCR showed expression patterns of the cyclin gene TTHERM_00180970 in Tetrahymena
thermophila. Peaks intensities were observed at C1, C6 and C13 with a recovery at C16 to the
near basal level intensity seen during vegetative growth.
427V 428V 427S 428S C0
C1
C2
C3
C4
C5
C6
C7
C8
C9 C10 C11
C12 C13 C14 C15
C16 C17
Figure 3: Gel electrophoresis of RT-PCR from 427 and 428 mating types undergoing conjugation during vegetation,
starvation and through 18 hours post mixing
4000
3500
Signal Intensity
3000
2500
2000
1500
1000
500
C18
C17
C16
C15
C14
C13
C12
C11
C10
C9
C8
C7
C6
C5
C4
C3
C2
C1
C0
428S
427S
428V
427V
0
RNA Collection Time Points
Figure 3: RT-PCR data of mRNA from 427 and 428 mating types undergoing conjugation during vegetation, starvation and
through 18 hours post mixing
Discussion:
The RT-PCR analysis of TTHERM_00180970 showed several additional peaks when compared
to the microarray data. Both the RT-PCR and microarray analysis showed a decrease in gene
expression upon starvation and a near zero expression during time 0 of conjugation. The RTPCR analysis had a sharp spike at C1 and began to diminish until time C3. There was another
small peak at C6, and intermediate peak at C13 followed by a decrease in intensity with a
rebound in gene expression at the end of conjugation at C17. The microarray data suggests that
gene expression is low at C0 with a small increase at C1. This is then followed by a decrease in
expression followed by a drastic increase in intensity at C8 with it diminishing to around normal
starvation levels at C10 and a rebound at the end of conjugation comparable to the vegetative
expression.
The discrepancy in expression patterns may be due to the fact that we extracted mRNA at
time points of every hour. The microarray data was obtained starting at C0 and following the
points ever two hours. Since the expression patterns seen form the RT-PCR data typically
increases sharply for a short time and diminishes quickly also; the full intensity of the peaks may
have been missed.
The expression patterns TTHERM_00180970 during conjugation suggests that the cyclin
gene may be involved in the stability of the macronucleus. The gene is on moderately in
vegetative growth which may be important normal stabilization of the macronucleus. Around
hour 6 we seen a drastic increase in gene expression through hour 9 followed by a sharp decrease
at hour 10. If this cyclin is involved in macronucleus stability, the sharp increase in gene
expression when the new macronucleuses are being formed. The sharp decrease at hour 10 may
signal for the degradation of one of the macronucleuses. Once the macronucleus is degraded
around hour 10 or 11 we see an increase in the amount of gene expression back to the normal
amount seen in vegetative growth which also supports that it may be involved in macronucleus
stability
References:
Miao W, Xiong J, Bowen J, Wang W, Liu Y, Braguinets O, Grigull J, Pearlman R, Orias E,
Gorovsky M. 2009. Microarray Analyses of Gene Expression during the Tetrahymena
thermophila Life Cycle. PloSONE; 4(2): e4429
TGED Search. http://tged.ihb.ac.cn/search.aspx?keyword=TTHERM_00180970. (Date
Accessed: 10/20/2009). Tetrahymena Gene Expression Database. TTHERM_00180970
Zhang H, Adl SM, Berger JD. 1999. Two distinct classes of mitotic cyclin homologues, Cyc1
and Cyc2, are involved in cell cycle regulation in the ciliate Paramecium tetraurelia.
Journal of Eukaryotic Microbiology. 46(6):585-96.